Novel estrogenic compounds

ABSTRACT

Novel estrogenic compounds of Formula I are provided.  
                 
 
     wherein the bond represented by the wavy line may be a single or double bond such that when the wavy line is a single bond, R 1  is selected from the group consisting of hydrogen, sulfate and glucoronate or other esters, and when the wavy line is a double bond, R 1  does not exist; R 2  is lower alkyl; R 3  may be selected from the group consisting of hydrogen, sulfate, or glucuronide or other esters; and R 4  through R 13  may independently be selected from the group consisting of hydrogen, hydroxy, ketone, lower alkyl, lower alkoxy, halogen, and carbonyl groups and R 14  is selected from the group consisting of hydrogen, sulfate and glucoronide and other esters. When R 1  is hydroxy, the hydroxy or ester substituent may have either an α or a β orientation. Compositions of matter including compounds of the present invention are also provided as are methods of treating mammals in need of treatment using compounds of the present invention.

CROSS REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional ApplicationNo. 60/188,523 filed Mar. 10, 2000, the disclosure of which is herebyincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

[0002] The present invention relates to the isolation of estrogeniccompounds.

BACKGROUND OF THE INVENTION

[0003] Women, particularly menopausal and post-menopausal women, oftenexperience a wide variety of conditions and disorders attributable toestrogen deprivation. Estrogen deprivation is most often the result ofloss of ovarian function. Exemplary conditions are hot flashes, drynessof the vagina, including discomfort during intercourse, loss of bonemass, increased heart disease and the like.

[0004] Providing dosages of estrogen is an effective agent for thecontrol or prevention of such conditions, particularly in controlling orpreventing hot flashes and vaginal atrophy, along with retarding orpreventing osteoporosis. Estrogen is typically administered alone or incombination with a progestin.

[0005] As detailed in U.S. Pat. No. Re. 36,247 to Plunkett et al.,estrogen alone, given in small doses, on a continuous basis, iseffective in most patients for the control of the above symptoms andproblems associated therewith. However, although the vast majority ofwomen taking continuous low-dose estrogen will not have bleeding formany months or even years, there is a distinct risk posed by thisroutine of silently (i.e. exhibiting no overt symptoms) developing“hyperplasia of the endometrium”. This term refers, of course, to anoverstimulation of the lining of the uterus which can becomepre-malignant, coupled with the possibility that the patient mayeventually develop cancer of the uterine lining even under such alow-dose regimen (Gusberg et al., Obstetrics and Gynaecology, 17,397-412, 1961).

[0006] Estrogen alone can also be given in cycles, usually 21-25 days ontreatment and 5-7 days off treatment. Again, if small doses of estrogenare required to control the symptoms and it is used to this fashion,only about 10% of women will experience withdrawal bleeding between thecycles of actual treatment. However, one must again be concerned by therisk of developing endometrial hyperplasia and by the increased relativerisk of developing cancer of the uterus (Research on the Menopause:Report of a W.H.O. Scientific Group, 53-68, 1981).

[0007] The addition of progestin for the last 7-10 days of each estrogencycle may virtually eliminate the concern about developing endometrialhyperplasia and/or also reduce the risk of developing endometrialcarcinoma below that of the untreated general population. However,withdrawal bleeding may occur regularly in this routine and this ishighly unacceptable to most older women (Whitehead, Am. J. Obs/Gyn.,142,6, 791-795, 1982).

[0008] Still another routine for estrogen administration may involve aformulation such as those found in birth control pills which containrelatively small doses of estrogen over the full 20-21 day treatmentcycle, plus very substantial doses of potent progestins over the sameperiod of time. This routine, of course not only produces withdrawalbleeding on each cycle, but is further unacceptable because suchformulations have been shown to carry an increased risk of developingarterial complications, such as stroke or myocardial infarction in olderwomen about the age of 35-40. This is especially true if the individualis a smoker of cigarettes (Plunkett, Am. J. Obs/Gyn. 142,6, 747-751,1982). There, however, remains a need for novel isolated estrogeniccompounds.

SUMMARY OF THE INVENTION

[0009] Thus, as one aspect of the present invention, a compoundrepresented by Formula I is provided.

[0010] where tie bond represented by the wavy line may be a single ordouble bond such that when the wavy line is a single bond, R₁ may beselected from the group consisting of hydrogen, sulfate and glucoronideor other esters, and when the wavy line is a double bond, R₁ does notexist; R₂ is lower alkyl; R₃ may be selected from the group consistingof hydrogen, sulfate, and glucuronide or other esters; and R₄ throughR₁₃ may independently be selected from the group consisting of hydrogen,hydroxy, ketone, lower alkyl (C₁ to C₄), lower alkoxy (C₁ to C₄),halogen, and carbonyl groups. When R₁ is hydroxy, the hydroxy or estersubstituent may have either an α or a β orientation, with the βorientation being preferred. R₂ is preferably C₁ to C₄ alkyl, and morepreferably is methyl. R₄ through R₁₂ are preferably hydrogen. R₁₃ ispreferably hydrogen or ethynyl. R₁₄ is hydrogen, sulfate, or glucoronidand other esters.

[0011] The compound represented by Formula I may be present inchemically pure form, namely greater than about 90% pure, preferablygreater than about 95% pure, and most preferably greater than about 99%pure.

[0012] A preferred compound is illustrated in Formula II:

[0013] Another preferred compound is illustrated in Formula III:

[0014] In another aspect, the present invention provides a compositionof matter. The composition of matter comprises a compound according tothe present invention.

[0015] In still another aspect, the invention provides a method oftreating mammals in need of treatment. The method comprisesadministering an effective amount of a composition of matter accordingto the present invention. Examples of treatments that are addressed bythe compositions of the invention include vasomotor symptoms, atrophicvaginitis, and osteoporosis.

[0016] The invention is described in greater detail with respect to thepreferred embodiments set forth hereinbelow.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The accompanying drawings which are incorporated in andconstitute a part of the specification, illustrate embodiments of theinvention and, together with the description, serve to explainprinciples of the invention.

[0018]FIG. 1 is a HPLC Chromatogram using chromatographic method 1showing Peaks A and B in the Endeavor 10-Component Conjugated Estrogensdrug product;

[0019]FIG. 2 is a HPLC Chromatogram using chromatographic method 1showing Peak A in the 17α-dihydroequilin sulfate, sodium salt standard;

[0020]FIG. 3 is a HPLC Chromatogram using chromatographic method 1showing Peak B in the Equilin sulfate, sodium salt standard;

[0021]FIG. 4 is a HPLC Chromatogram using chromatographic method 4showing re-injection of Peak A to determine its approximate purity;

[0022]FIG. 5 is a low resolution negative ion FAB-MS spectrum of Peak A;

[0023]FIG. 6 is a full range 400 MHz ¹H-NMR spectrum of Peak A ind₆-DMSO;

[0024]FIG. 7 is a 400 MHz ¹H-NMR spectrum of the aliphatic region ofPeak A in d₆-DMSO;

[0025]FIG. 8 is a 400 MHz ¹H-NMR spectrum of the aromatic region of PeakA in d₆-DMSO;

[0026]FIG. 9 is a full range 400 MHz 2D COSY ¹H-NMR spectrum of Peak Ain d₆-DMSO;

[0027]FIG. 10 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aliphaticregion of Peak A in d₆-DMSO;

[0028]FIG. 11 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aromaticregion of Peak A in d₆-DMSO;

[0029]FIG. 12 is a full range 100 MHz ¹³C-NMR spectrum of Peak A ind₆-DMSO;

[0030]FIG. 13 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region(0-40 ppm) of Peak A in d₆-DMSO;

[0031]FIG. 14 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region(40-80 ppm) of Peak A in d6-DMSO;

[0032]FIG. 15 is a 100 MHz ¹³C-NMR spectrum of the aromatic region ofPeak A in d₆-DMSO;

[0033]FIG. 16 is a full range 2D HMQC spectrum of the correlations ofthe protons and carbons of Peak A in d₆-DMSO;

[0034]FIG. 17 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons in Peak A in d₆-DMSO;

[0035]FIG. 18 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons (Zoom-in of FIG. 17) in Peak A in d₆-DMSO;

[0036]FIG. 19 is a 2D HMQC spectrum of the correlations of the aromaticprotons and carbons in Peak A in d₆-DMSO;

[0037]FIG. 20 is a full range 2D HMBC spectrum of the correlations ofthe protons and carbons in Peak A in d₆-DMSO;

[0038]FIG. 21 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbons in Peak A in d₆-DMSO;

[0039]FIG. 22 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and aromatic carbons in Peak A in d₆-DMSO;

[0040]FIG. 23 is a 2D HMBC spectrum of the correlations of the aromaticprotons and carbons in Peak A in d₆-DMSO;

[0041]FIG. 24 is a 2D HMBC spectrum of the correlations of the aromaticprotons and aliphatic carbons in Peak A in d₆-DMSO;

[0042]FIG. 25 is a HPLC Chromatogram using chromatographic method 4showing re-injection of Peak B to determine its approximate purity;

[0043]FIG. 26 is a low resolution negative ion FAB-MS spectrum of PeakB;

[0044]FIG. 27 is a full range 400 MHz ¹H-NMR spectrum of Peak B ind₆-DMSO;

[0045]FIG. 28 is a 400 MHz ¹H-NMR spectrum of the aliphatic region ofPeak B in d₆-DMSO;

[0046]FIG. 29 is a 400 MHz ¹H-NMR spectrum of the aromatic region ofPeak B in d₆-DMSO;

[0047]FIG. 30 is a full range 400 MHz 2D COSY ¹H-NMR spectrum of Peak Bin d₆-DMSO;

[0048]FIG. 31 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aliphaticregion of Peak B in d₆-DMSO;

[0049]FIG. 32 is a 400 MHz 2D COSY ¹H-NMR spectrum of the aromaticregion of Peak B in d₆-DMSO;

[0050]FIG. 33 is a full range 100 MHz ¹³C-NMR spectrum of Peak B ind₆-DMSO;

[0051]FIG. 34 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region(0-38 ppm) of Peak B in d₆-DMSO;

[0052]FIG. 35 is a 100 MHz ¹³C-NMR spectrum of the aliphatic region(43-50 ppm) of Peak B in d₆-DMSO;

[0053]FIG. 36 is a 100 MHz ¹³C-NMR spectrum of the aromatic region ofPeak B in d₆-DMSO;

[0054]FIG. 37 is a 100 MHz ¹³C-NMR spectrum of the carbonyl region ofPeak B in d₆-DMSO;

[0055]FIG. 38 is a full range 2D HMQC spectrum of the correlations ofthe protons and carbons of Peak B in d₆-DMSO;

[0056]FIG. 39 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons in Peak B in d₆-DMSO;

[0057]FIG. 40 is a 2D HMQC spectrum of the correlations of the aliphaticprotons and carbons (Zoom in of FIG. 39) in Peak B in d₆-DMSO;

[0058]FIG. 41 is a 2D HMQC spectrum of the correlations of the aromaticprotons and carbons in Peak B in d₆-DMSO;

[0059]FIG. 42 is a full range 2D HMBC spectrum of the correlations ofthe protons and carbons in Peak B in d₆-DMSO;

[0060]FIG. 43 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbons in Peak B in d₆-DMSO;

[0061]FIG. 44 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and aromatic carbons in Peak B in d₆-DMSO;

[0062]FIG. 45 is a 2D HMBC spectrum of the correlations of the aliphaticprotons and carbonyl carbons in Peak B in d₆-DMSO;

[0063]FIG. 46 is a 2D HMBC spectrum of the correlations of the aromaticprotons and carbons in Peak B in d₆-DMSO; and

[0064]FIG. 47 is a 2D HMBC spectrum of the correlations of the aromaticprotons and aliphatic carbons in Peak A in d₆-DMSO.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] The invention will now be described with reference to theembodiments set forth herein. These embodiments are intended toillustrate the invention and are not meant to limit the scope of theinvention, which is defined by the claims.

[0066] In one aspect of the present invention, a compound represented byFormula I is provided.

[0067] wherein the bond represented by the wavy line may be a single ordouble bond such that when the wavy line is a single bond, R₁ may beselected from the group consisting of hydrogen, sulfate and glucoronideor other esters, and when the wavy line is a double bond, R₁ does notexist; R₂ is lower alkyl; R₃ may be selected from the group consistingof hydrogen, sulfate and glucuronide or other esters; and R₄ through R₁₃may independently be selected from the group consisting of hydrogen,hydroxy, ketone, lower alkyl (C₁ to C₄), lower alkoxy (C₁ to C₄),halogen, and carbonyl groups. When R₁ is hydroxy, the hydroxy or estersubstituent may have either an α or a β orientation, with the βorientation being preferred. R₂ is preferably C₁ to C₄ alkyl, and morepreferably is methyl. R₄ through R₁₂ are preferably hydrogen. R₁₃ ispreferably hydrogen or ethynyl. R₁₄ may be selected from the groupconsisting of hydrogen, sulfate and glucoronide and other esters

[0068] The compound represented by Formula I is present in chemicallypure form, (i.e., greater than about 90% pure). The compound representedby Formula I is preferably greater than about 95% pure, and is mostpreferably greater than about 99% pure A preferred compound isillustrated in Formula II:

[0069] Another preferred compound is illustrated in Formula III:

[0070] Compounds of the present invention may be present in a conjugatedform. The conjugates may be various conjugates understood by thoseskilled in the art, including, but not limited to, glucuronide andsulfate. The most preferred conjugate is sulfate.

[0071] Compounds of the present invention may also be present as variouspharmaceutically acceptable salts including salts of the conjugatedcompound. The salts may be various salts understood by those skilled inthe art, including, but not limited to, sodium salts, calcium salts,magnesium salts, lithium salts, and amine salts such as piperazinesalts. The most preferred salts are sodium salts.

[0072] In another aspect, the present invention provides a compositionof matter. The composition of matter comprises one or more compoundsaccording to the present invention.

[0073] In one embodiment, the composition of the invention includes atleast one additional pharmaceutically active ingredient. Examples ofadditional active ingredients include, but are not limited to, otherestrogenic compounds, androgenic compounds, progestin compounds,vasodilation agents, calcium salts, and vitamin D and its derivatives(e.g., calcitriol and mixtures and blends thereof) and mixtures andblends of the various compounds.

[0074] Examples of estrogenic compounds and compositions are set forthin U.S. patent application Ser. No. 09/524,132 filed on Mar. 10, 2000,which is commonly assigned to the assignee of the present invention, thedisclosure of which is incorporated by reference herein in its entirety.Suitable estrogenic compounds include estrone, 17α-estradiol,17β-estradiol, equilin, 17α-dihydroequilin, 17β-dihydroequilin,equilenin, 17α-dihydroequilenin, 17β-dihydroequilenin,Δ^(8,9)-dehydroestrone, 17α-Δ^(8,9)-dehydroestradiol,17β-Δ^(8,9)-dehydroestradiol, ethinyl estradiol, estradiol valerate,6-OH equilenin, 6-OH 17α-dihydroequilenin, 6-OH 17β-dihydroequilenin,and mixtures, conjugates and salts thereof, and the estrogen ketones andtheir corresponding 17α- and 17β-hydroxy derivatives. The estrogeniccompounds may also be present as conjugated estrogens. The conjugatesmay be various conjugates understood by those skilled in the art,including, but not limited to, sulfate and glucuronide. The mostpreferred estrogen conjugates are estrogen sulfates. The estrogeniccompounds may also be present estrogen conjugates. In one embodiment,the estrogenic compounds are present as salts of estrogen conjugates.The salts may be various salts understood by those skilled in the art,including, but not limited to, sodium salts, calcium salts, magnesiumsalts, lithium salts, and piperazine salts. The most preferred salts aresodium salts. The estrogenic compounds can be derived from natural andsynthetic sources.

[0075] Suitable androgenic compounds include methyltestosterone,androsterone, androsterone acetate, androsterone propionate,androsterone benzoate, androsteronediol, androsteronediol-3-acetate,androsteronediol- 17-acetate, androsteronediol-3-17-diacetate,androsteronediol-17-benzoate, androsteronediol-3-acetate-17-benzoate,androsteronedione, dehydroepiandrosterone, sodium dehydroepiandrosteronesulfate, dromostanolone, dromostanolone propionate, ethylestrenol,fluoxymesterone, methyl testosterone, nandrolone phenpropionate,nandrolone decanoate, nandrolone furylpropionate, nandrolonecyclohexane-propionate, nandrolone benzoate, nandrolonecyclohexanecarboxylate, oxandrolone, oxymetholone, stanozolol,testosterone, testosterone decanoate, 4-dihydrotestosterone,5α-dihydrotestosterone, testolactone, 17α-methyl-19-nortestosterone andpharmaceutically acceptable esters and salts thereof, and combinationsof any of the foregoing.

[0076] Suitable vasodilation compounds include alpha andrenergicantagonists. Exemplary α-adrenergic compounds include phentolamine,phenoxybenzalamine, tolazoline, doxazosin, dibenamine, prazosin,prazosin hydrochloride, phenoxybenzamine and the like. Preferably,phentolamine is used and can form pharmaceutically acceptable salts withorganic and inorganic acids, as described, for example, in U.S. Pat. No.6,001,845 to Estok, the disclosure of which is incorporated herein byreference in its entirety. Preferably phentolamine mesylate orphentolamine hydrochloride is used. Other vasodilation compounds includephosphodiesterase type 5 inhibitors (e.g., suldenafi), prostaglandin Ecompounds (e.g., alprostodil), thymoxamine, bromocriptine, yohimbine,paperverine, apomorphine, organic nitrates, imipramine, verapamil,naftidrofuryl, and isoxsuprine. Combinations of the various vasodilationcompounds may be used.

[0077] Examples of progestins are set forth in U.S. Pat. No. Re. 36,247to Plunkett et al., the disclosure of which is incorporated herein byreference in its entirety. Suitable progestin compounds includedesogestrel, dydrogesterone, ethynodiol diacetate, medroxyprogesterone,levonorgestrel, medroxyprogesterone acetate, hydroxyprogesteronecaproate, norethindrone, norethindrone acetate, norethynodrel,allylestrenol, 19-nortestosterone, lynoestrenol, quingestanol acetate,medrogestone, norgestrienone, dimethisterone, ethisterone, cyproteroneacetate, chlormadinone acetate, megestrol acetate, norgestimate,norgestrel, desogestrel, trimegestone, gestodene, nomegestrol acetate,progesterone, 5α-pregnan-3β,20α-diol sulfate, 5α-pregnan-3β,20β-diolsulfate, 5α-pregnan-3β-ol-20-one, 16,5α-pregnen-3β-ol-20-one,4-pregnen-20β-ol-3-one-20-sulfate and mixtures thereof.

[0078] Calcium salts may include, without limitation, organic acid saltsof calcium such as calcium citrate, calcium lactate, calcium fumurate,calcium acetate, and calcium glycerophosphate, as well as inorganicsalts such as calcium chloride, calcium phosphate, calcium sulphate, andcalcium nitrate.

[0079] Pharmaceutically acceptable salts, solvates, hydrates, andpolymorphs may be formed of any of the active ingredients employed inthe composition of the invention. The invention also encompassesembodiments in which the composition of matter defmed herein is includedin various quantities in combination with known pharmaceuticallyaccepted formulations. For example, the composition of matter of theinvention may be incorporated into various known estrogen-containingdrug products such as, Premarin® made commercially available byWyeth-Ayerst Laboratories of Philadelphia, Pa. The composition of matterof the invention may also be employed as part of a continuousestrogen-progestin therapy regimen such as that described by U.S. Pat.No. Re. 36,247 to Plunkett et al. and made commercially available asPrempro® and Premphase® by Wyeth-Ayerst Laboratories.

[0080] The present invention also encompasses pharmaceuticallyacceptable drug products comprising a composition of matter of thepresent invention and at least one pharmaceutically acceptable carrier,diluent, or excipient, the selection of which are known to the skilledartisan. The drug product formulations can be in various forms such as,for example, tablets; effervescent tablets; pills; powders; elixirs;suspensions; emulsions; solutions; syrups; soft and hard gelatincapsules; transdermal patches; topical gels, creams and the like;suppositories; sterile injectable solutions; and sterile packagedpowders, sublingual tablets, buccal tablets, and buccal adhesivesystems.

[0081] In certain embodiments, the drug product is present in a solidpharmaceutical composition that may be suitable for oral administration.A solid composition of matter according to the present invention may beformed and may be mixed with an excipient, diluted by an excipient orenclosed within such a carrier which can be in the form of a capsule,sachet, tablet, paper, or other container. When the excipient serves asa diluent, it may be a solid, semi-solid, or liquid material which actsas a vehicle, carrier, or medium for the composition of matter.

[0082] Various suitable excipients will be understood by those skilledin the art and may be found in the National Formulary 19, pages2404-2406 (2000), the disclosure of pages 2404 to 2406 beingincorporated herein in their entirety. For example, the drug productformulations may include lubricating agents such as, for example, talc,magnesium stearate and mineral oil; wetting agents; emulsifying andsuspending agents; binding agents such as starches, gum arabic,microcrystalline cellulose, cellulose, methylcellulose, and syrup;anticaking agents such as calcium silicate; coating agents such asmethacrylates and shellac; preserving agents such as methyl- and propylhydroxybenzoates; sweetening agents; or flavoring agents. Polyols,buffers, and inert fillers may also be used. Examples of polyolsinclude, but are not limited to, mannitol, sorbitol, xylitol, sucrose,maltose, glucose, lactose, dextrose, and the like. Suitable buffersencompass, but are not limited to, phosphate, citrate, tartarate,succinate, and the like. Other inert fillers which may be used encompassthose which are known in the art and are usefull in the manufacture ofvarious dosage forms. If desired, the solid formulations may includeother components such as bulking agents and/or granulating agents, andthe like. The drug products of the invention may be formulated so as toprovide quick, sustained, or delayed release of the active ingredientafter administration to the patient by employing procedures well knownin the art.

[0083] To form tablets for oral administration, the composition ofmatter of the present invention may be made by a direct compressionprocess. In this process, the active drug ingredients may be mixed witha solid, pulverant carrier such as, for example, lactose, saccharose,sorbitol, mannitol, starch, amylopectin, cellulose derivatives orgelatin, and mixtures thereof, as well as with an antifriction agentsuch as, for example, magnesium stearate, calcium stearate, andpolyethylene glycol waxes. The mixture may then be pressed into tabletsusing a machine with the appropriate punches and dies to obtain thedesired tablet size. The operating parameters of the machine may beselected by the skilled artisan. Alternatively, tablets for oraladministration may be formed by a wet granulation process. Active drugingredients may be mixed with excipients and/or diluents. The solidsubstances may be ground or sieved to a desired particle size. A bindingagent may be added to the drug. The binding agent may be suspended andhomogenized in a suitable solvent. The active ingredient and auxiliaryagents may also be mixed with the binding agent solution The resultingdry mixture is moistened with the solution uniformly. The moisteningtypically causes the particles to aggregate slightly, and the resultingmass is pressed through a stainless steel sieve having a desired size.The mixture is then dried in controlled drying units for the determinedlength of time necessary to achieve a desired particle size andconsistency. The granules of the dried mixture are sieved to remove anypowder. To this mixture, disintegrating, antifriction, and/oranti-adhesive agents are added. Finally, the mixture is pressed intotablets using a machine with the appropriate punches and dies to obtainthe desired tablet size. The operating parameters of the machine may beselected by the skilled artisan.

[0084] If coated tablets are desired, the above-prepared cores may becoated with a concentrated solution of sugar or cellulosic polymers,which may contain gum arabic, gelatin, talc, titanium dioxide, or with alacquer dissolved in a volatile organic solvent, aqueous solvent, or amixture of solvents. To this coating, various dyes may be added in orderto distinguish among tablets with different active compounds or withdifferent amounts of the active compound present. In a particularembodiment, the active ingredient may be present in a core surrounded byone or more layers including enteric coating layers.

[0085] Soft gelatin capsules may be prepared in which capsules contain amixture of the active ingredient and vegetable oil. Hard gelatincapsules may contain granules of the active ingredient in combinationwith a solid, pulverulent carrier, such as, for example, lactose,saccharose, sorbitol, mannitol, potato starch, corn starch, amylopectin,cellulose derivatives, and/or gelatin.

[0086] In one preferred embodiment, the formulation is in the form oforally-administered tablets which contain the composition of matter ofthe present invention as set forth herein along with the followinginactive ingredients: calcium phosphate tribasic, calcium sulfate,carnauba wax, cellulose, glyceryl monooleate, lactose, magnesiumstearate, methylcellulose, pharmaceutical glaze, polyethylene glycol,stearic acid, sucrose, and titanium dioxide. Such ingredients may bepresent in amounts similar to those present in Premarin® (conjugatedestrogens tablets, USP) made commercially available by Wyeth-AyerstLaboratories of Philadelphia, Pa. Tablets employing the activeingredients of the invention may contain excipients similar to thosecontained in the 0.3 mg., 0.625 mg., and 1.25 mg tablets of Premarin®(conjugated estrogens tablets, USP).

[0087] Liquid preparations for oral administration may be prepared inthe form of syrups or suspensions, e.g., solutions containing an activeingredient, sugar, and a mixture of ethanol, water, glycerol, andpropylene glycol. If desired, such liquid preparations may containcoloring agents, flavoring agents, and saccharin. Thickening agents suchas carboxymethylcellulosemay also be used.

[0088] In the event that the above formulations are to be used forparenteral administration, such a formulation may comprise sterileaqueous injection solutions, non-aqueous injection solutions, or bothcomprising the composition of matter of the present invention. Whenaqueous injection solutions are prepared, the composition of matter maybe present as a water-soluble pharmaceutically acceptable salt.Parenteral preparations may contain anti-oxidants, buffers,bacteriostats, and solutes which render the formulation isotonic withthe blood of the intended recipient. Aqueous and non-aqueous sterilesuspensions may include suspending agents and thickening agents. Theformulations may be presented in unit-dose or multi-dose containers, forexample sealed ampules and vials. Extemporaneous injection solutions andsuspensions may be prepared from sterile powders, granules and tabletsof the kind previously described.

[0089] In a preferred embodiment, the drug product of the presentinvention is in the form of an injectable solution containing apredetermined amount (e.g., 25 mg) of the composition of matter in asterile lyphilized cake which also contains lactose, sodium citrate, andsimethicone. The pH of a solution containing the above ingredients maybe adjusted using a suitable buffer (e.g., sodium hydroxide orhydrochloric acid). Reconstitution may be carried out according to knownmethods, e.g., using a sterile diluent (5 mL) containing 2 percentbenzyl alcohol in sterile water. A preferred injectable solution issimilar to Premarin® Intravenous made commercially available byWyeth-Ayerst Laboratories.

[0090] The composition of matter also may be formulated such that it issuitable for topical administration (e.g., vaginal cream). Theseformulations may contain various excipients known to those skilled inthe art. Suitable excipients may include, but are not limited to, cetylesters wax, cetyl alcohol, white wax, glyceryl monostearate, propyleneglycol monostearate, methyl stearate, benzyl alcohol, sodium laurylsulfate, glycerin, mineral oil, water, carbomer, ethyl alcohol, acrylateadhesives, polyisobutylene adhesives, and silicone adhesives.

[0091] In a preferred embodiment, the drug product is in the form of avaginal cream containing the composition of matter as set forth hereinpresent in a nonliquefying base. The nonliquefying base may containvarious inactive ingredients such as, for example, cetyl esters wax,cetyl alcohol, white wax, glyceryl monostearate, propylene glycolmonostearate, methyl stearate, benzyl alcohol, sodium lauryl sulfate,glycerin, and mineral oil. Such composition may be formulated similar toPremarin® Vaginal Cream made commercially available by Wyeth-AyerstLaboratories.

[0092] Dosage units for rectal administration may be prepared in theform of suppositories which may contain the composition of matter in amixture with a neutral fat base, or they may be prepared in the form ofgelatin-rectal capsules which contain the active substance in a mixturewith a vegetable oil or paraffin oil.

[0093] In another aspect, the present invention relates to methods oftreating mammals (e.g., man) in need of treatment. The methods includeadministering an effective amount of a composition of matter as definedherein to the mammal in need of treatment. The methods may be used for anumber of treatments such as, but not limited to, vasomotor symptoms;atrophic vaginitis; osteoporosis; hypoestrogenism due to hypogonadism,castration, or primary ovarian failure; breast cancer in selectedpersons with metastatic disease; advanced androgen-dependent carcinomaof the prostate; abnormal uterine bleeding; and kraurosis vulvae. Theadministration may be cyclic, occurring for one or more short periods oftime or courses of treatment (i.e. short-term use). Alternatively, theadministration may be continuous, occurring over extended periods oftime (i.e. long-term use). One example of long-term use would be fromthe onset of menopause until death. Cyclic and continuous administrationmay be either uninterrupted or interrupted. Uninterrupted administrationoccurs one or more times daily such that there is no break in treatment.Interrupted administration occurs other than daily, for example arepeated course of treatment including three weeks of daily treatmentfollowed by one week of no treatment.

EXAMPLES

[0094] The present invention will now be described in greater detailwith respect to the following numbered examples. In the examples, “mL”means rmilliliter, “° C.” means degrees Ceicius, “mM” meansmillimoles/liter, “M” means moles/liter, “Å” means angstrom, “μm” meansmicrometer, “nm” means nanometer, “mm” means millimeter, “mg” meansmilligram, and “m/z” means mass to charge ratio. These examples are forillustrating the invention and are not intended to limit the inventionas set forth by the claims.

[0095] A list of instruments and equipment employed in the examples areas follows:

[0096] 1. HPLC Chromatographic Procedures

[0097] a. Analytical scale chromatographic system

[0098] 1. HP1100 Diode-array detector

[0099] 2. HP1100 Quaternary HPLC pump

[0100] 3. Shimadzu, Model RF-551, fluorescence detector

[0101] 4. HP1100 Thermostatically controlled column compartment

[0102] b. Semi-prep scale chromatographic system

[0103] 1. HP1100 HPLC chromatographic system

[0104] 2. HP1100 Diode-array detector

[0105] 3. HP1100 Quaternary HPLC pump

[0106] 4. HP1100 Thermostatically controlled column compartment

[0107] c. Prep scale chromatographic system

[0108] 1. Waters Delta Prep 4000 chromatographic system

[0109] 2. Waters 2487 UV detector

[0110] 3. Waters fraction collector II

[0111] 4. Waters PrepLC 40 mm radial compression assembly

[0112] 5. Waters Nova-Pak HR C₁₈ gun radial compression column segments,(2) 40 mm×100 mm segments with a 40 mm×10 mm guard segment

[0113] 2. Fraction Collection, Purification, and Crystallization

[0114] a. ISCO Foxy Jr., Fraction Collector

[0115] b. Büchi, Model R-124 rotary evaporator

[0116] c. Sep-Pak, SPE cartridges, Varian Bond Elut C₁₈

[0117] d. Waters fraction collector II

[0118] 3. Mass Spectral Analyses

[0119] a. Fast Atom Bombardment (FAB-MS)

[0120] 1. Instrument: VG Analytical ZAB 2-SE

[0121] 2. Sample input: Cesium ion gun

[0122] 3. Data system: VG Analytical 11-250J with PDP 11/73

[0123] 4. Solvent: Methanol

[0124] 5. Matrix: Glycerol/Thioglycerol/Triethylamine

[0125] b. High Resolution Mass Spectrometer (HR-MS)

[0126] 1. Instrument: VG Analytical ZAB 2-SE

[0127] 2. Sample input: Cesium ion gun

[0128] 3. Data System: VG Analytical 11-250J with PDP 11/73

[0129] 4. Solvant: Methanol

[0130] 5. Matrix: Peak A: PEG 300 & PEG 400 Peak B: PEG 300 &m-nitrobenzyl alcohol

[0131] B. Chemicals, Reagents, and Analytical Materials

[0132] 1. Chemicals and Reagents

[0133] a. Acetonitrile (ACN), HPLC grade

[0134] b. Methanol (MeOH), HPLC grade

[0135] c. Milli-Q water

[0136] d. Triethylamine (TEA), HPLC grade

[0137] e. tert-Butyl ammonium hydroxide (TBAH), 0.4 M, reagent grade

[0138] f. Potassium phosphate monobasic, AR grade

[0139] g. Nitrogen gas, zero grade

[0140] h. Phosphoric acid, 85%

[0141] i. Hydrochloric acid, concentrated

[0142] j. Sodium hydroxide

[0143] 2. Analytical Samples

[0144] Conjugated estrogens tablets, 1.25 mg, FDL lot #00426-064

[0145] 3. Analytical Standards

[0146] a. Conjugated Estrogens Reference Standard (ten component),Organics/LaGrange, Inc. (OLG) lot #C02322

[0147] b. Equilin sulfate, sodium salt, OLG lot#RD 1810

[0148] c. Equilin sulfate, sodium salt, Diosynth lot#00004429

[0149] d. 17α-Dihydroequilin sulfate, sodium salt, OLG lot# RD 1812

[0150] e. 17α-Dihydroequilin sulfate, sodium salt, Diosynth lot#58

[0151] f. Δ^(8,9)-Dehydroestrone, Proquina lot#9371-1/95

[0152] g. Δ^(8,9)-Dehydroestrone, lot# HS 30/95, supplier unknown

[0153] h. Δ^(8,9)-Dehydroestrone, sulfate +estrone sulfate, sodiumsalts, lot#1744, supplier unkown

Examples 1-4 Isolation of Compounds A and B

[0154] The compounds of Peaks A and B are isolated from a 10-componentconjugated estrogen available from Endeavor Pharmaceuticals ofWilmington, N.C. as follows:

Example 1 HPLC Chromatographic Assay Method 1 (Analytical Scale)

[0155] A standard solution containing about 0.03 mg/mL of ConjugatedEstrogens Drug Substance may be prepared. The drug substance may beprovided in powder form or a powder may be formed by grinding tablets.An appropriate amount of drug substance is weighed to yield 200 mL ofsolution. The drug substance is placed in a 200 ml volumetric flask. A61 mL volume of organic diluent is added to the flask and the flask ismechanically shaken for 15 minutes. About 100 mL of aqueous diluent isthen added to the flask and the flask is once again mechanically shakenfor 15 minutes. The resulting solution is diluted to volume with aqueousdiluent and mixed well. A portion of the solution is filtered through a0.45 μm PTFE filter.

[0156] A 50 mM phosphate buffer solution may be prepared using potassiumphosphate.

[0157] An aqueous diluent solution containing phosphate buffer and 0.4 MTBAH with a volumetric ratio of 277:0.9 may be prepared. The pH can beadjusted to 3.0±0.1 using phosphoric acid.

[0158] An organic diluent solution containing acetonitrile and methanolwith a volumetric ratio of 26.5:4 may be prepared.

[0159] A mobile phase may be prepared by mixing organic diluent andaqueous diluent to form a solution with a volumetric ratio of 30.5:69.5,organic:aqueous.

[0160] When 1.25 mg tablets are to be analyzed, five washed and groundtablets are placed into a 200 mL volumetric flask. A 61 mL volume oforganic diluent is added to the flask and the flask is mechanicallyshaken for 15 minutes. About 100 mL of aqueous diluent is then added tothe flask and the flask is once again mechanically shaken for 15minutes. The resulting solution is diluted to volume with aqueousdiluent and mixed. A portion of the solution is filtered through a 0.45μm PTFE filter.

[0161] In this chromatographic analysis, an HPLC system with a columnheater equipped with a 3 μm, 15.0 cm×4.6 mm C₁₈ column and suitable UVdetector for detection at 220 nm and diode array was employed. The flowrate was set for 1.5 mL/minute and the column temperature was set for25° C.

[0162] An example of a chromatographic procedure is as follows: Equalvolumes of the standard solution and the sample preparations wereseparately injected into the chromatographic systems. Peaks A and B wereintegrated and evaluated based on the peak area response for thechromatogram (area percent).

Example 2 HPLC Chromatographic Separation Method 2 (Semi-Prep Scale)

[0163] A Mobile Phase A (aqueous) containing 15 mM TEA in 0.125%concentrated HCl may be prepared by combining 42 mL of TEA with 20 L ofwater and mixing well. To the resulting solution, 25 mL of concentratedHCl is added and the pH is adjusted to approximately 7.0 with 1 N HCl or1 N NaOH.

[0164] A Mobile Phase B (organic) containing 15 mM TEA in 0.125%concentrated HCl in acetonitrile may be prepared by combining 16.8 mL ofTEA with 8 L of acetonitrile and adding 10 mL of concentrated HCl. Theresulting solution is then mixed well.

[0165] A sample for Peak A collection containing a 30 mg/mL solution of17α-dihydroequilin sulfate, sodium salt in mobile phase A may beprepared.

[0166] In this chromatographic separation for Peak A collection, asemi-prep scale HPLC system equipped with an appropriate fractioncollector, a Waters Symmetry C₁₈ (7.8 mm×300.0 mm), 7 μm column, and asuitable UV detector for detection at 220 nm may be employed. The flowrate may be set for 5 mL/minute, the column temperature may be set to40° C., and the gradient elution profile may be as follows: Time %Mobile Phase (Minutes) A B 0.0 83 17 42.0 83 17 42.1 50 50 47.0 50 5047.1 83 17

[0167] An example of a chromatographic procedure is as follows: 150 μLportions of the sample solution were separately injected into thechromatograph until all of the sample solution had been injected. Thefraction containing the peak at approximately 41 minutes was collectedand labeled as Peak A.

Example 3 HPLC Chromatographic Separation Method 3 (Prep Scale)

[0168] A Mobile Phase A (aqueous) containing 60 mM TEA in 0.5%concentrated HCl may be prepared by combining 168 mL of TEA with 20 L ofwater and mixing well. To the resulting solution, 100 mL of concentratedHCl is added and the pH is adjusted to approximately 3.0 with 1 N HCl or1 N NaOH.

[0169] A Mobile Phase B (organic) containing 60 mM TEA in 0.5%concentrated HCl in acetonitrile may be prepared by combining 84 mL ofTEA with 10 L of acetonitrile and adding 50 mL of concentrated HCl. Theresulting solution is then mixed well.

[0170] A sample solution for Peak A collection containing a 20 mg/mLsolution of 17α-dihydroequilin sulfate, sodium salt in mobile phase Amay be prepared.

[0171] A sample solution for Peak B collection containing a 20 mg/mLsolution of equilin sulfate, sodium salt in mobile phase A may beprepared.

[0172] In this chromatographic separation for Peak A collection, a prepscale HPLC system equipped with an appropriate fraction collector, two40 mm×100.0 mm radial compression C₁₈ column segments, a 40 mm×10 mmguard segment and a suitable UV detector for detection at 220 nm withthe full scale absorbance set at 4.0 may be employed. The flow rate maybe set for 50 mL/minute, the temperature is preferably ambienttemperature, and the gradient elution profile may be as follows: Time %Mobile Phase (Minutes) A B 0.0 83 17 43.0 83 17 43.1 40 60 52.0 40 6052.1 83 17

[0173] ln this chromatographic separation for Peak B collection, a prepscale HPLC system equipped with an appropriate fraction collector, two40 mm×100.0 mm radial compression C₁₈ column segments, a 40 mm×10 mmguard segment and a suitable UV detector for detection at 220 nm withthe full scale absorbance set at 4.0 may be employed. The flow rate maybe set for 50 mL/minute, the temperature is preferably ambienttemperature, and the gradient elution profile may be as follows: Time %Mobile Phase (Minutes) A B 0.0 80 20 35.0 80 20 35.1 40 60 41.0 40 6041.1 80 20

[0174] An example of a chromatographic procedure for Peak A collectionis as follows: 10 mL portions of the sample solution for Peak A wereseparately injected into the chromatograph until all of the samplesolution had been injected. The fraction containing the peak atapproximately 39 minutes was collected and labeled as Peak A.

[0175] An example of a chromatographic procedure for Peak B collectionis as follows: 10 mL portions of the sample solution for Peak B wereseparately injected into the chromatograph until all of the samplesolution had been injected. The fraction containing the peak atapproximately 29 minutes was collected and labeled as Peak B.

Example 4 HPLC Chromatographic Assay Method 4 (Analytical Scale)

[0176] A Mobile Phase A (aqueous) containing 15 mM TEA in 0.125%concentrated HCl may be prepared by combining 4.2 mL of TEA with 2 L ofwater and mixing well. To the resulting solution, 2.5 mL of concentratedHCl is added and the pH is adjusted to approximately 3.0 with 1 N HCl or1 N NaOH.

[0177] A Mobile Phase B (organic) containing 15 mM TEA in 0.125%concentrated HCl in acetonitrile may be prepared by combining 4.2 mL ofTEA with 2 L of acetonitrile and adding 2.5 mL of concentrated HCl. Theresulting solution is then mixed well.

[0178] A mobile phase may be prepared using a gradient system or bymanually preparing a mixture of 80% (v/v) Mobile Phase A and 20% (v/v)Mobile Phase B.

[0179] A sample solution containing approximately 0.06 mg/mL conjugatedestrogens in mobile phase may be prepared by transferring one 1.25 mgtablet into a 25 mL volumetric flask. A 6 mL volume of Mobile Phase B isadded to the flask and the flask is mechanically shaken for 10 minutes.The resulting solution is diluted to volume with Mobile Phase A and thenmixed well. A portion of the solution is filtered through a 0.45 μm PTFEfilter.

[0180] In this chromatographic analysis, an HP1100 HPLC system with acolumn heater equipped with a 3 μm, 15.0 cm×4.6 mm C₁₈ column andsuitable UV detector for detection at 220 nm and diode array wasemployed. The flow rate may be set for 1.5 mL/minute and the columntemperature may be set for 25° C.

[0181] An example of a chromatographic procedure is as follows: Equalvolumes of the standard solution and the sample preparations wereseparately injected into the chromatographic systems. Peaks A and B wereintegrated and evaluated based on the peak area response for thechromatogram (area percent).

Examples 5 and 6 Characterization of Peaks A and B

[0182] Examples 5 and 6 detail the characterization of Peaks A and Bfound in the Endeavor Pharmaceuticals 10-Component Conjugated Estrogensdrug product (FIG. 1). These compounds were also found to be present incertain estrogen standards which were evaluated. Peak A was found to bepresent in the 17α-dihydroequilin sulfate, sodium salt standard (FIG. 2)and Peak B was found to be present in the equilin sulfate, sodium saltstandards (FIG. 3). Due to the simplicity of the standard materials, theindividual peaks were collected by chromatographic fraction collectionfrom the corresponding standard and upon purification, were isolated asyellowish amorphous materials.

[0183] The fractioned samples were analyzed using techniques such asmass spectrometry (MS), and one-dimensional (1D) and two-dimensional(2D) nuclear magnetic resonance (NMR) spectrometry. Mass spectrometryutilized both low and high resolution fast atom bombardment massspectrometry (FAB-MS) to determine the accurate molecular weight andempirical formula of the compounds. For the NMR analyses, samples weredissolved in deuterated dimethyl sulfoxide (d₆-DMSO), which permittedthe hydroxyl protons to be visible during analysis. NMR techniquesincluded ¹H-NMR (proton), ¹³C-NMR (carbon), homonuclear correlationspectroscopy (COSY), distortionless enhancement by polarization transfer(DEPT), heteronuclear multiple quantum coherence (HMQC), andheteronuclear multiple bond correlation (HMBC). 2D COSY analyses helpeddetermine the correlation of neighboring protons to one another, whileDEPT analyses determined the assignment of carbon type. 2D HMQC analysesprovided information about what protons are attached to what carbons,and 2D HMBC analyses showed longer range coupling of the proton andcarbon atoms (usually 2 to 4 bonds away).

Example 5 Peak A Characterization

[0184] Separation and Isolation

[0185] Peak A was isolated as a triethyl ammonium salt due to theion-pairing agent of the mobile phase of the HPLC chromatographic methoddescribed above in Example 2. After the fraction was collected, most ofthe ACN was removed by rotary evaporation, and the fraction was furtherconcentrated using a C₁₈ SPE cartridge, washed with water, and elutedwith approximately 10 mL of methanol. The fraction was then brought todryness under a.stream of dry nitrogen. Using the HPLC method describedin above Example 4, a small portion of the 5.3 mg of Peak A isolated fortesting by MS and NMR was redissolved in mobile phase and injected intothe HPLC system to determine the purity of the fraction. This injectionof Peak A showed a purity of about 89% (FIG. 4).

[0186] Mass Spectral Analyses

[0187] Preliminary negative ion FAB-MS spectral data of the isolatedfraction of Peak A indicated that the molecular weight was approximately363 m/z (FIG. 5). A negative ion HR-MS study indicated a mass of363.0909 amu that compares well with the calculated mass of 363.0902 amufor the proposed molecular formula of C₁₈H₁₉O₆S₁ for Peak A.

[0188] Proton (¹H) and 2D COSY Nuclear Magnetic Resonance Spectroscopy

[0189] The ¹H-NMR and the 2D COSY spectra of Peak A in deuterateddimethyl sulfoxide (d₆-DMSO) are shown in FIGS. 6-8 and 9-11,respectively. The peak assignments, based upon the proton NMR spectraand COSY spectral couplings, are shown in Table 1 and are consistentwith the proposed structure of Peak A.

[0190] Carbon (¹³C), 2D HMQC, and 2D HMBC Nuclear Magnetic Resonance

[0191] The ¹³C-NN, HMQC, and HMBC spectra of Peak A in deuterateddimethyl sulfoxide (d₆-DMSO) are shown in FIGS. 12-15, 16-19, and 20-24,respectively. In order to collect the data more quickly and with agreater signal to noise ratio, the ¹³C-NMR spectrum was obtainednon-quantitatively, and integrations were not performed. Peakassignments based upon the carbon NMR, HMQC, and HMBC spectralinterpretations are shown in Table 2 and were consistent with theproposed structure of Peak A. 2D HMBC data can be more difficult tointerpret, since it is possible that all crosspeaks are not observed.HMBC signals may typically occur with H—C connectivities that are 2 to 4bonds removed, but also can detect some 1 to 2 bond connections. TABLE 1Summary Table of Proton NMR and COSY Band Assignments Chemical ShiftTenative Number of COSY (ppm) Multiplicty* Protons Couplings**Assignment 0.52 s 3 12b 18 1.17 t 9 19 20 1.52 m 1 14, 15b, 16a, 16b 15a1.60 m 1 15a, 15b, 16b  6a 1.73 m 1 11a, 11b, 12b 12a 2.04 m 1 11a, 11b,12a, 18 12b 2.16 m 1 14, 15a, 16a 15b 2.27 m 1 15a, 16a, 17 16b 2.50 — —— solvent-DMSO 2.99 m 1 12a, 12b, 11b 11a 3.09 q 6 20 19 3.10 d 1 15a,15b 14 3.13 m 1 12a, 12b 11b 3.17 — — — solvent-MeOH 3.33 — — —solvent-H₂O 3.76 t 1 16b, 17-OH 17 4.09 — — — solvent-MeOH 4.50 d(w) 117 17(OH) 6.62 s 1 —  7 7.30 d of d 1  1, 4  2 7.75 d 1  2  1 7.85 d(w)1  2  4 8.90 bs 1 — NH⁺ 9.64 s 1 —  6(OH)

[0192] TABLE 2 Summary Table of Carbon NMR, HMQC, and HMBC PeakAssignments Chemical Shift Tenative Number Of HMQC HMBC (ppm) CarbonsCouplings Couplings Assignments  8.6 3 1.17 — 20  15.8 1 0.52 12, 13, 1718  23.4 1 2.99, 3.13  8, 9, 13 11  24.6 1 1.52, 2.16  8, 13, 16 15 29.2 1 1.73, 2.04  9, 11, 13, 18 12  32.9 1 1.60, 2.27 15, 17 16 39-40— — — solvent- DMSO  44.4 1 — — 13  44.5 1 3.10  8, 9, 12, 13, 15, 18 14 45.7 1 3.09 — 19  48.5 — — — solvent- MeOH  77.3 1 3.76 13, 15, 18 17107.5 1 6.62  5, 6, 9, 14  7 111.7 1 7.85  2, 3, 6, 10  4 119.9 1 — —  9121.5 1 7.30 10  2 123.6 1 7.75  3, 5, 9, 10  1 123.7 1 — —  5 129.4 1 —— 10 136.0 1 — —  8 149.4 1 — —  3 150.7 1 — —  6 — — 4.50 13, 16, 1717(OH) — — 9.64  5, 6, 7  6(OH)

[0193] 1D and 2D NMR Spectral Interpretation

[0194] Peak A is a derivative of dihydroequilenin, which contains fivearomatic protons and ten aliphatic protons. The ¹H-NMR spectrum exhibitsthe ten expected aliphatic protons, but only four main signals wereobserved in the aromatic region (6.5-8.0 ppm) of the ¹H-NMR (FIG. 8)that corresponded to a 1:1:1:1 ratio. Based upon the splitting expectedfrom the proposed structure for Peak A, these signals were consistentwith a dihydroequilenin based ring structure substituted at one of thearomatic protons. The four aromatic ¹H-NMR signals showed a strongsinglet and a strong doublet, and a second singlet and doublet, whichare weakly split into doublets.

[0195] Substitution at each of the possible aromatic positions maycreate a distinct splitting pattern. Substitution at the 1-position maycreate a single pair of strong doublets (H6 and H7) with a strong COSYcorrelation and a pair of singlets (H4 and H2) which may exhibit a weakCOSY correlation and be weakly split by each other. Substitution at the2-position may create but a single pair of strong doublets (H6 and H7)with a strong COSY correlation and a pair of singlets (H4 and H1).Substitution of the aromatic ring system at the 4-position may create apattern of 2 strong pairs of doublets with strong COSY correlations inthe spectrum. Substitution at the 6- or 7-position may create but asingle pair of strong doublets (HI and H2) with a strong COSYcorrelation and a pair of singlets (H4 and H6 or H7). The H4 proton maybe expected to interact weakly with the H2 proton exhibiting a weak COSYcorrelation and causing the H2 doublet and the H4 singlet to be weaklysplit by each other.

[0196] Based upon the splitting pattern of the aromatic protons,substitution of the hydroxyl group in Peak A may be at either the H6 orH7 position. The pair of doublets at 7.75 and 7.30 ppm for H1 and H2,respectively, is shown to be adjacent from the 2D COSY spectrum (FIG.11). H2 and H4 at 7.85 ppm exhibited a weak COSY correlation that causedH2 to appear as a doublet of doublets due to splitting by both H1 andH4; and H4 as a strong singlet weakly split to a doublet. Theassignments of the corresponding carbons C1, C2, and C4 were based uponthe HMQC spectra (FIG. 19) at 123.6, 121.5, and 111.7 ppm, respectively.The other aromatic ring contains only one proton at 6.62 ppm for H6 orH7, which was observed as a singlet as expected since no other protonsare nearby to cause splitting. The corresponding carbon signal wasassigned from the HMQC correlations at 107.5 ppm (FIG. 19).

[0197] In the ¹³C-NMR aromatic region (100-170 ppm) (FIG. 15), therewere four large signals and six smaller signals. Protonated carbons maytypically have larger signals than non-protonated carbons and that wasused in differentiating among the aromatic carbon atoms. This wasverified by observation of only four HMQC signals in this region, whichoccur only for carbons with directly attached protons, in the aromaticregion (FIG. 19). The remaining six aromatic carbon signals did not haveHMQC peaks, and are therefore, non-protonated.

[0198] Two of the six non-protonated signals are shifted downfield toabout 150 ppm (149.4 and 150.7 ppm), which may be typical of aromaticcarbon atoms attached to an oxygen atom. This fits the proposedstructure with the normal 3-position hydroxy sulfate ester and theproposed hydroxyl substitution on an aromatic position. The HMBCspectrum (FIG. 23) shows correlations of the carbon signals at 149.4 ppmto H1 and H4 and the carbon signal at 150.7 ppm to H4 and either H6 orH7. Based upon those correlations the signal at 149.4 ppm must be C3.The signal at 150.7 ppm must be C6 or the H4 correlation would have beena weak 4-bond correlation. Thus, the substitution is at the 6-positionand the aromatic proton at 6.62 ppm and the aromatic carbon at 107.5 ppmare assigned H7 and C7, respectively. The remaining four non-protonatedcarbon atoms (119.9, 123.7, 129.4, and 136.0 ppm) match the number ofbridging non-protonated carbon atoms expected for the proposedstructure. Assignment of these four signals can be made from HMBCcorrelations (FIG. 23). H2 shows a single strong 3-bond correlation tothe carbon signal at 129.4 ppm and is assigned C10 being the onlybridging carbon atom within 3 bonds of H2. H1 exhibits three strong3-bond correlations at 119.9, 123.7, and 149.4 (C3) ppm and one weak2-bond correlation to 129.4 (C 10) ppm. H4 exhibits HMBC correlations to121.5 (C2), 129.4 (C10), 149.4 (C3), and 150.7 (C6) ppm. H7 exhibitsHMBC correlations to aromatic signals at 119.9, 123.7, and 150.7 (C6)ppm. Based upon these correlations, the carbon signals at 119.9 and123.7 ppm are for C5 and C9, but their exact assignments are not yetestablished.

[0199] There is a single strong signal downfield in the ¹H-NMR spectrumat 9.64 ppm (FIG. 8). This region is typical of aromatic phenolicprotons and this signal is assigned as H6(OH). HMBC spectrum for thisproton exhibits correlations at 107.5 (C7), 123.7, and 150.7 (C6) ppm(FIG. 23). Based upon these correlations and the relationships of theother protons the signal at 123.7 ppm must be C5 and thus the signal at119.9 ppm must be C9. This leaves only the aromatic carbon signal at136.0 ppm unassigned. Thus, the remaining aromatic signal by process ofelimination was assigned as C8.

[0200] The methyl region (0.5 to 1.5 ppm) of the ¹H-NMR spectrum (FIG.7) shows a strong methyl signal split into a triplet at 1.17 ppm that isindicative of the methyl proton (H20) of the triethyl amrmonium cation.This signal shows a strong COSY correlation to the quartet signal at3.09 ppm for the protons (H19) of the methylene group (FIG. 10). TheHMQC correlation spectrum (FIG. 17) showed corresponding carbon atoms at8.6 and 45.7 ppm for C20 and C19, respectively. The amine proton (NH⁺)of the cation was expected to have a ¹H-NMR chemical shift of about 8.0to 9.5 ppm; however, amines have the problem of slow exchange and oftenare not seen, or are only seen as a small broad peak in this region. TheNH proton in the ¹H-NMR spectrum was observed as a single broad signalat about 8.90 ppm for this compound (FIG. 8).

[0201] In the ¹³C-NMR aliphatic region (0-100 ppm) (FIGS. 13-14), therewere two strong signals at 8.6 and 45.7 ppm for the triethyl ammoniumcation, and eight signals for the aliphatic carbons of Peak A. Theproposed structure for Peak A contains eight aliphatic carbon atoms. Theeight aliphatic carbon signals were observed at 15.8, 23.4, 24.6, 29.2,32.9, 44.4, 44.5, and 77.3 ppm. Seven of the eight signals show HMQCcorrelations to proton signals (FIGS. 17-18). Only the carbon signal at44.4 ppm did not exhibit a correlation to any proton signal and wasconsidered a bridging carbon. The proposed structure has one bridgingaliphatic carbon atom, and thus the peak at 44.4 ppm was assigned asC13. The three carbon signals at 15.8, 44.5, and 77.3 ppm eachcorrelated to a single proton signal whereas the other four carbonsignals observed at 23.4, 24.6, 29.2, and 32.9 ppm each correlated totwo proton signals. This may be the case in saturated aliphatic ringsystems since the two protons of the methylene groups are present indiffering electronic environments and thus, exhibit different chemicalshifts.

[0202] Inspection of the three carbon signals with a single proton HMQCcorrelation shows that the signal at 15.8 ppm is in the expected methylregion for ¹³C-NMR and correlates by HMQC to the proton signal at 0.52ppm. These protons exhibited the expected integration ratio for a methylgroup of 3:1 relative to the individual aromatic protons and areassigned as H18 based on the chemical shift and the HMQC correlationsand is the only methyl group in the proposed structure for Peak A. Thesignal for the carbon atom (C17) attached to the hydroxyl group isexpected to shift downfield relative to the other aliphatic signals, asdescribed for the aromatic carbon signals. Thus, the carbon signalobserved at 77.3 ppm was assigned as C17 based upon chemical shift andthe HMQC correlated proton signal at 3.76 ppm was assigned as H17. Thereis only one remaining carbon atom with one proton, thus, the signal at44.5 ppm was assigned as C14 and the proton signal at 3.10 ppmcorrelated to it by HMQC was H14.

[0203] The remaining four aliphatic carbon signals each exhibited twoHMQC correlations to proton signals. The carbon signal at 23.4 ppmcorrelates to the protons at 2.99 and 3.13 ppm. The COSY spectrum (FIG.10) of these two protons show that they couple to the proton signals at1.73 and 2.04 ppm indicating the two sets are adjacent. The HMQCspectrum (FIG. 17) shows these protons both correlate to the carbonsignal at 29.2 ppm. The carbon signal at 24.6 ppm correlates to theprotons at 1.52 and 2.16 ppm. The COSY spectrum of these two protonsshow that they couple to the proton signals at 1.60 and 2.27 ppm,indicating the two sets are adjacent. The HMQC spectrum shows theseprotons both correlate to the carbon signal at 32.9 ppm. Theseobservations are consistent with the proposed structure of Peak A thathas two sets of adjacent methylene groups at C11 and C12, and at C15 andC16.

[0204] HMBC couplings (FIGS. 21-22) can be used to ascertain theidentity and position of each of the four methylene groups. The bridgingcarbon at 119.9 (C9) ppm correlates to only the protons of, the carbonsignals at 23.4 and 29.2 ppm (FIG. 22). This observation verifies thatthe carbon signals at 23.4 and 29.2 ppm must be assigned as C11 and C12and that the other two carbon signals at 24.6 and 32.9 ppm must beassigned as C15 and C16, but their exact assignments have not yet beenestablished. The proton at 0.52 (H18) ppm shows a EMBC correlation tothe carbon signals at 29.2, 44.4 (C13), and 77.3 (C17) ppm (FIG. 21).Based upon this, the carbon signal at 29.2 ppm was assigned as C12 andthus, the carbon signal at 23.4 ppm must be C11. Based upon the HMQCcorrelations (FIG. 18) of each of these carbon signals, the protonsignals at 2.99 and 3.13 ppm can be assigned as H11a and H12b,respectively, and the protons at 1.73 and 2.04 ppm can be assigned asH12a and H12b, respectively. The proton at 3.76 (C17) ppm shows COSYcorrelations to the protons at 2.27 ppm and 4.50 ppm (FIG. 10). Basedupon the COSY correlations of the proton signal at 2.27, it must beadjacent to H17 and thus the carbon signal at 32.9 ppm must be C16 andthe carbon signal at 24.6 ppm must be C15. Based upon the HMQCcorrelations (FIG. 18) of each of the carbon signals, the proton signalsat 1.52 and 2.16 ppm can be assigned as H15a and H15b, respectively, andthe protons at 1.60 and 2.27 ppm can be assigned as H16a and H16b,respectively. The proton at 4.50 ppm adjacent to the 3.76 (C17) ppmproton must be H17(OH), which would be expect to shift downfield beingdirectly attached to an oxygen atom. HMBC correlations of the proton at4.50 ppm (H17(OH)) exhibit correlations to 32.9 (C16), 44.4 (C13), and77.3 (C17) ppm carbon signals (FIG. 21). Other HMBC correlations aredetailed in Table 2 above and are consistent with the proposed structureof Peak A.

[0205] Additional signals in the proton NMR spectrum (FIGS. 7-8) wereobserved from water at 3.33 ppm, DMSO at 2.50 ppm, a small amount ofmethanol at 3.17 and 4.09 ppm, and other assorted small, unidentifiedaromatic and aliphatic “impurity” signals.

[0206] In addition to the expected aromatic and aliphatic signals ofPeak A, there are two other significant HMQC signals (FIG. 17). Thesolvent (d₆-DMSO) was observed at 39.5 ppm (HMQC peak at 2.50 ppm), andthe methanol was observed at 48.5 ppm (HMQC peak at 3.17 ppm).

[0207] Based on the observed NMR data the structure of Peak Acorresponds well to the proposed structure.

Example 6 Peak B Characterization

[0208] Separation and Isolation

[0209] Peak B was isolated as a triethyl ammonium salt due to theion-pairing agent of the mobile phase of the HPLC chromatographic methoddescribed above in Example 3. After the fraction was collected, most ofthe ACN was removed by rotary evaporation, and the fraction was furtherconcentrated using a C₁₈ SPE cartridge, washed with water, and elutedwith approximately 10 mL of methanol. The fraction was then brought todryness under a stream of dry nitrogen. Using the HPLC method describedabove in Example 4, a small portion of the 20.2 mg of Peak B isolatedfor testing by MS and NMR was redissolved in mobile phase and injectedon the HPLC system to determine the purity of the fraction. Thisinjection of Peak B showed a purity of about 82% (FIG. 25).

[0210] Mass Spectral Analyses

[0211] Preliminary negative ion FAB-MS spectral data of the isolatedfraction of Peak B indicated that the molecular weight was approximately361 m/z (FIG. 26). A negative ion HR-MS study indicated a mass of361.0744 amu that compares well with the calculated mass of 361.0746 amufor the proposed molecular formula of C₁₈H₁₇O₆S₁ for Peak B.

[0212] Proton (¹H) and 2D COSY Nuclear Magnetic Resonance Spectroscopy

[0213] The ¹H-NMR and the 2D COSY spectra of Peak B in deuterateddimethyl sulfoxide (d6-DMSO) are shown in FIGS. 27-29 and 30-32,respectively. The peak assignments, based upon the proton NMR spectraand COSY spectral couplings, are shown in Table 3 and are fullyconsistent with the structure of Peak B.

[0214] Carbon (¹³C), 2D HMQC, and 2D HMBC Nuclear Magnetic Resonance

[0215] The ¹³C-NMR, HMQC, and HMBC spectra of Peak B in deuterateddimethyl sulfoxide (d₆-DMSO) are shown in FIGS. 33-37, 38-41, and 42-47,respectively. In order to collect the data more quickly and with agreater signal to noise ratio, the ¹³C-NMR spectrun was obtainednon-quantitatively, and integrations were not performed. Peakassignments based upon the carbon NMR, HMQC, and HMBC spectralinterpretations are shown in Table 4, and are fully consistent with theproposed structure of Peak B. 2D HMBC data can be more difficult tointerpret, since it is possible that all crosspeaks are not observed.HMBC signals typically occur with H—C connectivities that are 2 to 4bonds removed, but also can detect some 1 to 2 bond connections. TABLE 3Summary Table of Proton NMR and COSY Band Assignments Chemical ShiftTenative Number of COSY (ppm) Multiplicty* Protons Couplings**Assignment 0.70 s 3 — 18 1.17 t 9 19 20 1.78 m 1 11b, 12b 12a 1.88 m 114, 15b, 16b 15a 2.00 m 1 11a, 12a 12b 2.32 m 1 16b 16a 2.39 m 1 14, 15a15b 2.50 — — — solvent-DMSO 2.62 m 1 15a, 16a 16b 3.08 m 1 12b 11a 3.10q 6 20 19 3.12 m 1 12a 11b 3.14 d 1 15a, 15b 14 3.18 — — — solvent-MeOH3.34 — — — solvent-H₂O 4.10 — — — solvent-MeOH 6.69 s 1 —  7 7.33 d of d1  1, 4  2 7.77 d 1  2  1 7.89 d(w) 1  2  4 8.88 bs 1 — NH⁺ 9.83 s 1 — 6(OH)

[0216] TABLE 4 Summary Table of Carbon NMR, HMQC, and HMBC PeakAssignments Chemical Shift Tenative Number Of HMQC HMBC (ppm) CarbonsCouplings Couplings Assignments  8.6 3 1.17 — 20  12.7 1 0.70 12, 13,14, 17 18  21.4 1 1.88, 2.39 13, 14, 17 15  22.9 1 3.08, 3.12  8, 9, 12,13 11  28.8 1 1.78, 2.00  9, 11, 13, 14 12  36.1 1 2.32, 2.62 14, 15, 1716 39-40 — — — solvent- DMSO  45.7 1 3.10 — 19  45.9 1 3.14  8, 9, 12,13, 15, 18 14  46.9 3 — — 13  48.5 — — — solvent- MeOH 106.0 1 6.69  4,5, 6, 9, 14  7 111.7 1 7.89  2, 3, 6, 10  4 120.1 1 — —  9 121.8 1 7.33 3, 4, 10  2 123.7 1 7.77  3, 4, 5, 6, 9, 10  1 124.2 1 — —  5 129.4 1 —— 10 133.3 1 — —  8 149.7 1 — —  3 151.2 1 — —  6 218.9 1 — — 17 — —9.83  5, 6, 7, 10  6(OH)

[0217] ID and 2D NMR Spectral Interpretation

[0218] Peak B is a derivative of equilenin, which contains five aromaticprotons and ten aliphatic protons. The ¹H-NMR spectrum exhibits the tenexpected aliphatic protons, but only four main signals were observed inthe aromatic region (6.5-8.0 ppm) of the ¹H-NMR (FIG. 29) thatcorresponded to a 1:1:1:1 ratio. Based upon the splitting expected fromthe proposed structure for Peak B, these signals were consistent with anequilenin based ring structure substituted at one of the aromaticprotons. The four aromatic ¹H-NMR signals showed a strong singlet and astrong doublet, and a second singlet and doublet, which are weakly splitinto doublets.

[0219] Substitution at each of the possible aromatic positions wouldcreate a distinct splitting pattern. Substitution at the 1-positionwould create a single pair of strong doublets (H6 and H7) with a strongCOSY correlation and a pair of singlets (H4 and H2) which would exhibita weak COSY correlation and be weakly split by each other. Substitutionat the 2-position would create but a single pair of strong doublets (H6and H7) with a strong COSY correlation and a pair of singlets (H4 andH1). Substitution of the aromatic ring system at the 4-position wouldcreate a pattern of 2 strong pairs of doublets with strong COSYcorrelations in the spectrum. Substitution at the 6- or 7-position wouldcreate but a single pair of strong doublets (H1 and H2) with a strongCOSY correlation and a pair of singlets (H4 and H6 or H7). The H4 protonwould be expected to interact weakly with the H2 proton exhibiting aweak COSY correlation and causing the H2 doublet and the H4 singlet tobe weakly split by each other.

[0220] Based upon the splitting pattern of the aromatic protons,substitution of the hydroxyl group in Peak B must be at either the H6 orH7 position. The pair of doublets at 7.77 and 7.33 ppm for H1 and H2,respectively, is shown to be adjacent from the 2D COSY spectrum (FIG.32). H2 and H4 at 7.89 ppm exhibited a weak COSY correlation that causedH2 to appear as a doublet of doublets due to splitting by both H1 andH4; and H4 as a strong singlet weakly split to a doublet. Theassignments of the corresponding carbons C1, C2, and C4 were based uponthe HMQC spectra (FIG. 41) at 123.7, 121.8, and 111.7 ppm, respectively.The other aromatic ring contains only one proton at 6.69 ppm for H6 orH7, which was observed as a singlet as expected since no other protonsare nearby to cause splitting. The corresponding carbon signal wasassigned from the HMQC correlations at 106.0 ppm (FIG. 41).

[0221] In the ¹³C-NMR aromatic region (100-170 ppm) (FIG. 36), therewere four large signals and six smaller signals. Protonated carbonstypically have larger signals than non-protonated carbons and that wasused in differentiating among the aromatic carbon atoms. This wasverified by observation of only four HMQC signals in this region, whichoccur only for carbons with directly attached protons, in the aromaticregion (FIG. 41). The remaining six aromatic carbon signals did not haveHMQC peaks, and are therefore, non-protonated.

[0222] Two of the six non-protonated signals are shifted downfield toabout 150 ppm (149.7 and 151.2 ppm), which is typical of aromatic carbonatoms attached to an oxygen atom. This fits the proposed structure withthe normal 3-position hydroxy sulfate ester and the proposed hydroxylsubstitution on an aromatic position. The HMBC spectrum (FIG. 46) showsstrong correlations of the carbon signals at 149.7 ppm to H1 and H4 andthe carbon signal at 151.2 ppm to H4 and either H6 or H7. Based uponthose correlations the signal at 149.7 ppm must be C3. The signal at151.2 ppm must be C6 or the H4 correlation would have been a weak 4-bondcorrelation. Thus, the substitution is at the 6-position and thearomatic proton at 6.69 ppm and the aromatic carbon at 106.0 ppm areassigned H7 and C7, respectively. The remaining four non-protonatedcarbon atoms (120.1, 124.2, 129.4, and 133.3 ppm) match the number ofbridging non-protonated carbon atoms expected for the proposedstructure. Assignment of these four signals can be made from HMBCcorrelations (FIG. 46). H2 showed correlations to the carbon signals at111.7 (C4), 129.4, and 149.7 (C3) ppm. C10 was the only bridging carbonatom within 3 bonds of H2 and was assigned to the carbon signal at 129.4ppm. H1 exhibits correlations at 111.7 (C4), 120.1, 124.2, 129.4 (C10),149.7 (C3), and 151.2 (C6) ppm. H4 exhibits HMBC correlations to 121.8(02), 129.4 (C10), 149.7 (C3), and 151.2 (C6) ppm. H7 exhibits HMBCcorrelations to aromatic signals at 111.7 (C4), 120.1, 124.2, and 151.2(C6) ppm. Based upon these correlations, the carbon signals at 120.1 and124.2 ppm must correspond to C5 and C9, but their exact assignments arenot yet established.

[0223] There is a single strong signal downfield in the ¹H-NMR spectrumat 9.83 ppm (FIG. 29). This region is typical of aromatic phenolicprotons and this signal is assigned as H6(OH). HMBC spectrum for thisproton exhibits correlations at 106.0 (C7), 124.2, 129.4 (C10), and151.2 (C6) ppm (FIG. 46). Based upon these correlations and therelationships of the other protons the signal at 124.2 ppm must be C5and thus the signal at 120.1 ppm must be C9. This leaves only thearomatic carbon signal at 133.3 ppm unassigned. Thus, the remainingaromatic signal by process of elimination was assigned as C8.

[0224] The methyl region (0.5 to 1.5 ppm) of the ¹H-NMR spectrum (FIG.28) shows a strong methyl signal split into a triplet at 1.17 ppm thatis indicative of the methyl proton (H20) of the triethyl ammoniumcation. This signal shows a strong COSY correlation to the quartetsignal at 3.10 ppm for the protons (H19) of the methylene group (FIG.31). The HMQC correlation spectrum (FIG. 39) showed corresponding carbonatoms at 8.6 and 45.7 ppm for C20 and C19, respectively. The amineproton (NH⁺) of the cation was expected to have a ¹H-NMR chemical shiftof about 8.0 to 9.5 ppm; however, amines have the problem of slowexchange and often are not seen, or are only seen as a small broad peakin this region. The NH proton in the ¹H-NMR spectrum was observed as asingle broad signal at about 8.88 ppm for this compound (FIG. 29).

[0225] In the ¹³C-NMR aliphatic region (0-100 ppm) (FIGS. 34-35), therewere two strong signals at 8.6 and 45.7 ppm for the triethyl ammoniumcation, and seven signals for the aliphatic carbons of Peak B. Theproposed structure for Peak B contains seven aliphatic carbon atoms anda ketone carbon. The seven aliphatic carbon signals were observed at12.7, 21.4, 22.9, 28.8, 36.1, 45.9, and 46.9 ppm. Carbonyl carbon atomsare known to shift far downfield to above 200 ppm. FIG. 37 showed such asignal present at 218.9 ppm and was assigned C17. Six of the sevensignals show HMQC correlations to proton signals (FIGS. 39-40). Only thecarbon signal at 46.9 ppm did not exhibit a correlation to any protonsignal and was considered a bridging carbon. The proposed structure hasone bridging aliphatic carbon atom, and thus the peak at 46.9 ppm wasassigned as C13. The carbon signals at 12.7 and 45.9 ppm each correlatedto a single proton signal, whereas the other four carbon signalsobserved at 21.4, 22.9, 28.8, and 36.1 ppm each correlated to two protonsignals. This is often the case in saturated aliphatic ring systemssince the two protons of the methylene groups are present in differingelectronic environments and thus, exhibit different chemical shifts.

[0226] Inspection of the two carbon signals with a single proton HMQCcorrelation shows that the signal at 12.7 ppm is in the expected methylregion for ¹³C-NMR and correlates by HMQC to the proton signal at 0.70ppm. These protons at 0.70 ppm exhibited the expected integration ratiofor a methyl group of 3:1 relative to the individual aromatic protonsand are assigned as H18 based on the chemical shift and the HMQCcorrelations and is the only methyl group in the proposed structure forPeak B. The remaining carbon atom with one proton was observed at 45.9ppm was assigned as C14. The proton signal at 3.14 ppm correlated to itby HMQC and was assigned H14.

[0227] The remaining four aliphatic carbon signals each exhibited twoHMQC correlations to proton signals. The carbon signal at 22.9 ppmcorrelates to the protons at 3.08 and 3.12 ppm. The COSY spectrum ofthese two protons show that they couple to the proton signals at 1.78and 2.00 ppm indicating the two sets are adjacent. The HMQC spectrumshows these protons both correlate to the carbon signal at 28.8 ppm. Thecarbon signal at 21.4 ppm correlates to the protons at 1.88 and 2.39ppm. The COSY spectrum of these two protons show that they couple to theproton signals at 2.32 and 2.62 ppm, indicating the two sets areadjacent. The HMQC spectrum shows these protons both correlate to thecarbon signal at 36.1 ppm. These observations are consistent with theproposed structure of Peak B that has two sets of adjacent methylenegroups at C11 and C12, and at C15 and C16.

[0228] HMBC couplings (FIGS. 43-44) can be used to ascertain theidentity and position of each of the four methylene groups. The bridgingcarbon at 120.1 (09) ppm correlates to only the protons of the carbonsignals at 22.9 and 28.8 ppm (FIG. 44). This observation verifies thatthe carbon signals at 22.9 and 28.8 ppm must be assigned as C11 and C12and that the other two carbon signals at 21.4 and 36.1 ppm must beassigned as C15 and C16, but their exact assignments are not yetestablished. The proton at 0.70 (H18) ppm shows a HMBC correlation tothe carbon signals at 28.8, 45.9 (014), 46.9 (C13), and 218.9 (C17) ppm(FIG. 43). Based upon this, the carbon signal at 28.8 ppm was assignedas C12 and thus, the carbon signal at 22.9 ppm must be C11. Based uponthe HMQC correlations (FIG. 40) of each of these carbon signals, theproton signals at 3.08 and 3.12 ppm can be assigned as H11a and H11b,respectively, and the protons at 1.78 and 2.00 ppm can be assigned asH12a and H12b, respectively. The proton at 3.14(014) ppm shows COSYcorrelations to the protons at 1.88 and 2.39 ppm (FIG. 31). Based uponthese COSY correlations of the proton signal at 2.14 ppm, it must beadjacent to H14 and thus the carbon signal at 21.4 ppm must be C15 andthe carbon signal at 36.1 ppm must be C16. Based upon the HMQCcorrelations (FIG. 40) of each of the carbon signals, the proton signalsat 1.88 and 2.39 ppm can be assigned as H15a and H15b, respectively, andthe protons at 2.32 and 2.62 ppm can be assigned as H16a and H16b,respectively. Other HMBC correlations are detailed in Table 4 above andare consistent with the proposed structure of Peak B.

[0229] Additional signals in the proton NMR spectrum (FIGS. 28-29) wereobserved from water at 3.34 ppm, DMSO at 2.50 ppm, a small amount ofmethanol at 3.18 and 4.10 ppm, and other assorted small, unidentifiedaromatic and aliphatic “impurity” signals. In addition to the expectedaromatic and aliphatic signals of Peak B (FIG. 38), methanol wasobserved at 48.5 ppm (HMQC peak at 3.18 ppm).

[0230] Based on the observed NMR data the structure of Peak Bcorresponds well to the proposed structure.

[0231] The present invention has been described herein with reference toits preferred embodiments. The embodiments do not serve to limit theinvention, but are set forth for illustrative purposes. The scope of theinvention is defined by the claims that follow.

That which is claimed:
 1. A compound represented by Formula I:

wherein: the bond represented by the wavy line may be a single or doublebond such that when the wavy line is a single bond, R₁ is selected fromthe group consisting of hydrogen, sulfate and glucroronate or otheresters, and when the wavy line is a double bond, R₁ does not exist; R₂is lower alkyl; R₃ is selected from the group consisting of hydrogen,sulfate, and glucuronide or other esters; R₄ through R₁₃ areindependently selected from the group consisting of hydrogen, hydroxy,ketone, lower alkyl, lower alkoxy, halogen, and carbonyl groups; and R₁₄is selected from the group consisting of hydrogen, sulfate andglucoronide or other esters; said compound being present in chemicallypure form.
 2. The compound according to claim 1, wherein said compoundis of Formula II:


3. The compound according to claim 1, wherein said compound is ofFormula III:


4. The compound according to claim 1, wherein said compound is greaterthan about 95% pure.
 5. The compound according to claim 1, wherein R₂ isC₁ to C₄ alkyl, R₄-R₁₂ are hydrogen and R₁₃ is hydrogen or ethynyl. 6.The compound according to claim 1, wherein when R₁ is hydroxy, thecompound has a β orientation.
 7. The compound according to claim 1 inconjugated form.
 8. The compound according to claim 1 having thefollowing physicochemical properties: molecular formula of C₁₈H₁₉O₆S;¹H-NMR spectrum as shown in FIG. 6; and ¹³C-NMR spectrum as shown inFIG.
 12. 9. The compound according to claim 1 having the followingphysicochemical properties: molecular formula of C₁₈H₁₇O₆S; ¹H-NMRspectrum as shown in FIG. 27; and ¹³C-NMR spectrum as shown in FIG. 33.10. A pharmaceutical composition incorporating a compound represented byFormula I:

wherein: the bond represented by the wavy line may be a single or doublebond such that when the wavy line is a single bond, R₁ is selected fromthe group consisting of hydrogen, sulfate and glucoronate and otheresters, and when the wavy line is a double bond, R₁ does not exist; R₂is lower alkyl; R₃ is selected from the group consisting of hydrogen,sulfate, and glucuronide or other esters; R₄ through R₁₃ areindependently selected from the group consisting of hydrogen, hydroxy,ketone, lower alkyl, lower alkoxy, halogen, and carbonyl groups; and R₁₄is selected from the group consisting of hydrogen, sulfate andglucoronide and other esters; said compound being present in chemicallypure form.
 11. The pharmaceutical composition according to claim 10,wherein said compound is of Formula II:


12. The pharmaceutical composition according to claim 10, wherein saidcompound is of Formula III:


13. The pharmaceutical composition according to claim 10, wherein saidcompound is greater than about 95% pure.
 14. The pharmaceuticalcomposition according to claim 10, wherein R₂ is C₁ to C₄ alkyl, R₄-R₁₂are hydrogen and R₁₃ is hydrogen or ethynyl.
 15. The pharmaceuticalcomposition according to claim 10, wherein when R₁ is hydroxy, thecompound has a B orientation.
 16. The pharmaceutical compositionaccording to claim 10, wherein said compound is in conjugated form. 17.The pharmaceutical composition according to claim 10, wherein thecomposition further comprises at least one additional pharmaceuticallyactive ingredient.
 18. The pharmaceutical composition according to claim17, wherein the at least one additional pharmaceutically activeingredient is selected from the group consisting of estrogeniccompounds, androgenic compounds, progestin compounds, vasodilationagents, calcium salts, and vitamin D and its derivatives, and mixturesand combinations thereof.
 19. The pharmaceutical composition accordingto claim 10, wherein said compound has the following physicochemicalproperties: molecular formula of C₁₈H₁₉O₆S; ¹H-NMR spectrum as shown inFIG. 6; and ¹³C-NMR spectrum as shown in FIG.
 12. 20. The pharmaceuticalcomposition according to claim 10, wherein said compound has thefollowing physicochemical properties: molecular formula of C₁₈H₁₇O₆S;¹H-NMR spectrum as shown in FIG. 27; and ¹³C-N spectrum as shown in FIG.33.
 21. A method of treating mammals in need of treatment, said methodcomprising administering an effective amount of a compound representedby Formula I:

wherein: the bond represented by the wavy line may be a single or doublebond such that when the wavy line is a single bond, R₁ is selected fromthe group consisting of hydrogen, sulfate and glucoronate or otheresters, and when the wavy line is a double bond, R₁ does not exist; R₂is lower alkyl; R₃ is selected from the group consisting of hydrogen,sulfate, and glucuronide or other esters; R₄ through R₁₃ areindependently selected from the group consisting of hydrogen, hydroxy,ketone, lower alkyl, lower alkoxy, halogen, and carbonyl groups; and R₁₄is selected from the group consisting of hydrogen, sulfate andglucoronide and other esters; said compound being present in chemicallypure form.
 22. The method according to claim 21, wherein said compoundis of Formula II:


23. The method according to claim 21, wherein said compound is ofFormula III:


24. The method according to claim 21, wherein said compound is greaterthan about 95% pure.
 25. The method according to claim 21, wherein R₂ isC₁ to C₄ alkyl, R₄-R₁₂ are hydrogen and R₁₃ is hydrogen or ethynyl. 26.The method according to claim 21, wherein when R₁ is hydroxy, thecompound has a β orientation.
 27. The method according to claim 21,wherein said compound is in conjugated form.
 28. The method according toclaim 21, wherein said compound is administered as part of apharmaceutical composition, said composition further comprising at leastone additional pharmaceutically active ingredient.
 29. The methodaccording to claim 28, wherein the at least one additionalpharmaceutically active ingredient is selected from the group consistingof estrogenic compounds, androgenic compounds, progestin compounds,vasodilation agents, calcium salts, and vitamin D and its derivatives,and mixtures and combinations thereof.
 30. The method according to claim21, wherein said compound has the following physicochemical properties:molecular formula of C₁₈H₁₉O₆S; ¹H-NMR spectrum as shown in FIG. 6; and¹³C-NMR spectrum as shown in FIG.
 12. 31. The method according to claim21, wherein said compound has the following physicochemical properties:molecular formula of C₁₈H₁₇O₆S; ¹H-NMR spectrum as shown in FIG. 27; and¹³C-NMR spectrum as shown in FIG. 33.